Compositions and Methods for Assaying hERG Channel Binding

ABSTRACT

The present invention provides compositions and methods useful for assaying binding of compounds to the hERG K +  channel. According to a method of the present invention, a compound of interest is added to the hERG K +  channel in the presence of a selenium analog of a competitive inhibitor of the hERG K +  channel. Next, the amount of the selenium analog of the competitive inhibitor that bound to the hERG K +  channel is quantified using mass spectrometry. The quantified amount can then be used to determine the amount of the compound of interest that bound to the hERG K +  channel. A selenium analog of any competitive inhibitor of the hERG K +  channel may be used according to the present invention, including but not limited to selenium analogs of the small molecule dofetilide; the peptide BeKm-1; or a combination of both.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/433,227, filed May 11, 2006, U.S. Pat. No. 7,326,772, issued Feb. 5,2008, which is incorporated herein by reference. U.S. patent applicationSer. No. 11/433,227 claims priority from U.S. Provisional PatentApplication No. 60/680,954, filed May 12, 2005, and U.S. ProvisionalPatent Application No. 60/695,103, filed Jun. 28, 2005, both of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to drug discovery. Moreparticularly, the present invention relates to compositions and methodsuseful for assaying binding of compounds to the hERG (HumanEther-a-go-go Related Gene) K⁺ channel.

BACKGROUND

Voltage-dependant ion channels are proteins that span cell surfacemembranes in excitable tissue such as heart and nerves. Ions passingthrough channels form the basis of the cardiac action potential. Influxof Na⁺ and Ca²⁺ ions, respectively, control the depolarizing upstrokeand plateau phases of the action potential. K⁺ ion efflux repolarizesthe cell membrane, terminates the action potential, and allowsrelaxation of the muscle. A rapid component of the repolarizing currentflows through the K+ channel encoded by the human ether-a-go-go-relatedgene (hERG). Impaired repolarization can prolong the duration of theaction potential, delay relaxation and promote disturbances of theheartbeat. Action potential prolongation is detected clinically as alengthening of the QT interval measured on the electrocardiogram (ECG).

Drug-induced QT prolongation is a serious complication of drugs due toimpaired repolarization, which is associated with an increased risk oflethal ventricular arrhythmias. Drug-induced QT prolongation is almostalways associated with block of the hERG K⁺ channel. A plethora ofdrugs, such as methanesulfonanilides, dofetilide, MK-499, and E-4031 areknown to block K⁺ ion channels such as hERG on the heart causing a lifethreatening ventricular arrhythmia and heart attack in susceptibleindividuals. Unfortunately, incidence of drug-induced ventriculararrhythmia is often too low to be detected in clinical trials.

A sudden death due to the blocking of hERG channels by noncardiovasculardrugs such as terfenadine (antihistamine), astemizole (antihistamine),and cisapride (gastrokinetic) led to their withdrawal from the market.Recently, drugs like Vioxx, Celebrex and Bextra were also pulled out ofthe market for concerns relating to dangerous cardiac side effects.Consequently, cardiac safety relating to K⁺ channels has become a majorconcern of regulatory agencies. In order to prevent costly attrition, ithas therefore become a high priority in drug discovery to screen outinhibitory activity on hERG channels in lead compounds as early aspossible.

Current methods for testing potential drug molecules for hERG blockingactivity have several limitations. Technologies based on cell-basedpatch clamp electrophysiology or animal tests are technically difficultand do not meet the demand for throughput and precision for preclinicalcardiac safety tests. Other assays use radio-labeled, fluorescent,dye-conjugated, or biotinylated markers for detection and quantificationof binding. However, many of these markers have reduced activity afterlabeling. In addition, the use of radio-labeled analogs poses practicallimitations such as requirements for complex infrastructure and licensesfor operating radioactive compounds. Accordingly, there is a need in theart to develop new compositions and methods for quantifying the bindingof drug molecules to hERG channels.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful forassaying binding of compounds to the hERG K⁺ channel. According to amethod of the present invention, a compound of interest is added to thehERG K⁺ channel in the presence of a selenium analog of a competitiveinhibitor of the hERG K⁺ channel. Next, the amount of the seleniumanalog of the competitive inhibitor that bound to the hERG K⁺ channel isquantified using mass spectrometry. The quantified amount can then beused to determine the amount of the compound of interest that bound tothe hERG K⁺ channel.

A selenium analog of any competitive inhibitor of the hERG K⁺ channelmay be used according to the present invention, including but notlimited to selenium analogs of the small molecule dofetilide; thepeptide BeKm-1, the sequence of which is shown in SEQ ID NO: 1; or acombination of both. BeKm-1 is a 36 amino acid peptide isolated from theCentral Asian scorpion Buthus eupeus that strongly binds to the hERG K⁺channel. Preferably, the selenium analog of the competitive inhibitorhas a K_(i) within an order of magnitude of the K_(i) of an unmodifiedversion of the competitive inhibitor, more preferably within about 3times the K_(i) of an unmodified version of the competitive inhibitor.

In a preferred embodiment, the amount of competitive inhibitor thatbound to the hERG K⁺ channel is quantified by quantifying the amount ofselenium that bound to the hERG K⁺ channel. This can be accomplishedusing any type of mass spectrometry, including but not limited to matrixassisted laser desorption time of flight (MALDI-TOF) mass spectrometry,electrospray mass spectrometry, and inductively coupled plasma massspectrometry (ICP-MS).

The present invention also provides selenium analogs of competitiveinhibitors of the hERG K⁺ channel. In one embodiment, at least onecysteine of BeKm-1 is replaced with seleno-cysteine. In one aspect ofthis embodiment, the seventh residue, cysteine, of BeKm-1 is replacedwith seleno-cysteine to form (SeC)⁷-BeKm-1. In another embodiment,methyl-seleno-cysteine is covalently bonded to the first amino acid ofBeKm-1 to form (MeSeC)⁰-BeKm-1. In yet another embodiment, the seleniumanalog is di-seleno-dofetilide. Preferably, the selenium analogs ofBeKm-1 have K_(i)s for the hERG K⁺ channel in the range of about 0.08 nMto about 1.0 nM. Also preferably, di-seleno-dofetilide has a K_(i) forthe hERG K⁺ channel in the range of about 4 nM to about 25 nM. Thepresent invention also includes pharmaceutically acceptable salts of theabove selenium analogs, including but not limited to acetate, TFA,maleate and hydrochloride salts.

BRIEF DESCRIPTION OF THE FIGURES

The present invention together with its objectives and advantages willbe understood by reading the following description in conjunction withthe drawings, in which:

FIG. 1 shows a schematic of synthesis of (SeC)⁷-BeKm-1 according to thepresent invention.

FIG. 2 shows a schematic of synthesis of (MeSeC)⁰-BeKm-1 according tothe present invention.

FIG. 3 shows a schematic of synthesis of di-seleno-dofetilide accordingto the present invention.

FIG. 4 shows the results of FlashBlue competition assays for BeKm-1analogs according to the present invention.

FIG. 5 shows detection of selenium in (SeC)⁷-BeKm-1 by mass spectrometryaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of detecting binding of acompound of interest to the hERG K⁺ channel. In a first step, a seleniumanalog of a competitive inhibitor of the hERG K⁺ channel is added alongwith the compound of interest to the hERG K⁺ channel. The hERG K⁺channel may be presented to the compounds in any form, including but notlimited to as purified protein on a protein chip, expressed on thesurface of cells, or from membranes prepared from hERG K⁺channel-expressing cells. In the latter two cases, the hERG K⁺ channelcould be presented in, for example, a 96-well plate format. According tothe present method, bound competitive inhibitor is quantified using massspectrometry (MS). This is possible by using the mass spectrometer todetect the presence and amount of selenium. Selenium is an element notfound in the hERG K⁺ channel. Thus, as long as the compound of interestdoes not contain selenium, the amount of bound competitive inhibitor canbe directly related to the amount of selenium present in the mixture.

The assay may be used to quantify the amount of selenium in two basicways. With the first method, a solution of the compound of interest anda solution of the competitive inhibitor would be added to the hERG K⁺channel and incubated for an appropriate period of time. At the end ofthe incubation period, the supernatant would be transferred from theassay and diluted into an appropriate volume. The diluted supernatantwould then be injected into a mass spectrometer, selenium would bedetected, and these values would be used to determine the amounts of thecompetitive inhibitor and the compound of interest that bound to thehERG K⁺ channel. With this method, an increase of selenium concentrationin the supernatant compared to assays with competitive inhibitor only(i.e. without any additional compound of interest) would correspond to areduction of binding of the competitive inhibitor to the hERG K⁺channel, and would indicate binding of the compound of interest to thehERG K⁺ channel.

With the second method, at the end of the incubation period, the hERG K⁺channel would be washed to remove unbound material and bound competitiveinhibitor would be released using, e.g., an appropriate denaturant. Thedenatured mixture would then be diluted in an appropriate volume andinjected into a mass spectrometer. Selenium would be detected and thesevalues would be used to determine the amount of competitive inhibitorbound and the amount of the compound of interest that bound to the hERGK⁺ channel. With this method, a decrease in selenium concentrationcompared to assays with competitive inhibitor alone would correspond toa decrease in bound competitive inhibitor, indicating that the compoundof interest bound the HERG K⁺ channel. With either method, the peak areaof the selenium-containing daughter ion in the absence of any compoundof interest would serve as the control selenium value.

Daughter ions may be produced from the mixture for MS analysis inseveral different ways, including but not limited to using electrosprayionization, matrix-assisted laser desorption/ionization (MALDI), andinductively coupled plasma (ICP) sources. The daughter ions may then beseparated using any type of MS, including but not limited totime-of-flight (TOF) MS. Selenium-containing daughter ions can then bedetected due to their unique mass-to-charge ratios. ICP-MS isparticularly well suited to this method, as it specifically detectsmetals such as selenium at a very low concentration.

The competitive inhibitor may be a selenium analog of any molecule thatbinds to the hERG K⁺ channel. Examples include selenium analogs of theBeKm-1 peptide and dofetilide and combinations thereof. Preferably, theselenium analog of the competitive inhibitor has a K_(i) within an orderof magnitude of the K_(i) of an unmodified version of the competitiveinhibitor, more preferably within about 3 times the K_(i) of theunmodified version. This will ensure adequate sensitivity of the assay.

In one embodiment, the competitive inhibitor is a selenium analog of thepeptide BeKm-1. The native amino acid sequence of BeKm-1 is RPTDI KCSESYQCFP VCKSR FGKTN GRCVN GFCDCF (SEQ ID NO: 1). In one aspect of thisembodiment, at least one cysteine in the native sequence is replacedwith seleno-cysteine. In one example, referred to hereafter as(SeC)⁷-BeKm-1, the seventh residue of the BeKm-1 peptide, a cysteine, isreplaced with seleno-cysteine. This modified peptide is well suited forthis assay as selenium is not found in the hERG receptor, and is notlikely to be found in the molecule of interest. Thus, the only source ofselenium would be from the competitive inhibitor. In addition, seleniumis chemically similar to sulfur, forming an S—Se bond similar to adisulfide bond, resulting in minimal changes to the structure of thenative BeKm-1 peptide.

(SeC)⁷-BeKm-1 can be made with solid phase synthesis techniques usingBoc-Phe-PAM (Butyloxycarbonyl-Phenylalanine-Phenylacetamidomethyl)polystyrene resin and Boc (Butyloxycarbonyl)-protected amino acidderivatives (FIG. 1). First, Boc-Phe-PAM is deprotected by removal ofthe Boc group (step 110). Next, after neutralization and checking of theamine bond, Boc-(pMB)Cys, the Boc derivative of the next amino acid inthe peptide sequence, is coupled to Phe-PAM (step 120). The amine bondis then checked and Boc-(pMB)Cys is recoupled if necessary. This cycleis then repeated with each amino acid derivative corresponding to thesequence in backward order until residue 8 (Ser) is reached (step 130).Boc-(pMB)Se-Cys is then coupled to the peptide and deprotected (step140) and synthesis is completed through the first residue in the peptide(Arg) (step 150). The completed peptide is then cleaved from the resinusing HF (step 160). The HF is then removed, the resin is extracted with50% ACN/H2O (0.1% TFA) and the peptide is diluted to <1 mM. The pH ofthe peptide solution is then adjusted to between about 7 and 7.5,folding is performed overnight, with folding monitored by high pressureliquid chromatography (HPLC), and the peptide is purified using HPLC(step 170).

In another aspect of this embodiment, methyl-seleno-cysteine is attachedto the N-terminal Arg residue of BeKm-1 to produce (MeSeC)⁰-BeKm-1.(MeSeC)⁰-BeKm-1 can be made using solid phase synthesis techniques,again using Boc-Phe-PAM polystyrene resin and Boc-protected amino acidderivatives (FIG. 2). In this case, the peptide is synthesized all theway up to the first residue, Arg (step 210). Boc-Methyl-Se-Cys is thencoupled to the peptide (step 220) and deprotected (step 230). Thecompleted peptide is then cleaved from the resin with HF (step 240) andthe peptide is diluted to <1 mM. The HF is then removed, the resin isextracted with 50% ACN/H2O (0.1% TFA) and the peptide is diluted to <1mM. The pH of the peptide solution is then adjusted to between about 7and 7.5, folding is performed overnight, with folding monitored by highpressure liquid chromatography (HPLC), and the peptide is purified usingHPLC (step 250).

In another embodiment, the competitive inhibitor is a selenium analog ofdofetilide(N-[4-(2-[2-[4-(methanesulphonamido)phenoxy]-N-methylethylamino]ethyl)phenyl]methane-sulfphonamide). In one aspect of this embodiment, the seleniumanalog is di-seleno-dofetilide(N-[4-(2-[2-[4-(methaneselenodoioxamido)phenoxyl]-N-methylethylamino)ethyl)phenyl]-methane-selenodioxamide).Di-seleno-dofetilide may be synthesized according to the scheme shown inFIG. 3, which is based on the synthesis of dofetilide described in U.S.Pat. No. 6,124,363. First, N-methyl-2-(4-nitrophenyl)ethylaminehydrochloride (compound 310) is mixed with4-(2-chloroethoxy)nitrobenzene (compound 320), anhydrous potassiumcarbonate, potassium iodide, tetra-n-butylammonium iodide and deionizedwater and heated under reflux for 3 hours. Next, the mixture is cooledto about 40° C., ethyl acetate is added, and the mixture is extractedwith ethyl acetate and concentrated. Ethanol is then added to theextract to precipitate the product and the product is filtered to giveN-Methyl-N-[2-(4-nitrophenoxy)ethyl]-4-nitrophenethylamine (compound330). In the next step, compound 330 and 5% w/v palladium-on-carbon areadded to methanol, stirred and hydrogenated at 60 p.s.i. to giveN-Methyl-N-[2-(4-aminophenoxy)ethyl]-4-aminophethylamine (compound 340).Compound 340 is then added to acetonitrile, followed by triethylamineand methaneselenodioxy chloride. Sodium carbonate is then added to thereaction mixture and distilled. Next, sodium hydroxide is added to themixture, followed by concentrated hydrochloric acid. The solid is thecollected by filtration, washed with water, and dried to obtaindi-seleno-dofetilide (compound 350).

BeKm-1 is thought to interact mostly with closed channels and block K⁺current by interacting with the hERG channel's outer vestibule. Incontrast, dofetilide is believed to interact with open and/orinactivated hERG channels, accessing the channel's vestibule from theinside of the cell. Thus, in one embodiment, selenium analogs of bothBeKm-1 and dofetilide are used to assay a compound of interest for itsability to bind to the hERG channel. The two analogs could be used inseparate experiments or combined into one experiment.

EXAMPLES Synthesis of Selenium Analogs of BeKm-1 Materials and Methods

Boc-Phenylalanyl-O-methyl-PAM resin (Loading: 0.67 mmoles of Boc-Phe/gresin) was bought from Peninsula Labs (Bachem, Inc.). Most Boc aminoacids, including Boc-S (pMB)-Cysteine, HOBT (N-hydroxybenzotriazole) andHBTU (2-[1H-Benzotriazole-1-yl]-1,1,3,3-tetramethylaminiumhexafluorophosphate) were bought from Peninsula Labs. Boc-Asparagine(Xan)-OH and Boc-Glutamine (Xan)-OH were bought from EMD Biosciences.SelenoCystine.2 HCl and Methyl Seleno-Cysteine were bought from SabinsaCorporation and were converted to Boc-(Se-pMB)-Cysteine andBoc-Methyl-Seleno-cysteine respectively. Ethyldiisopropyl amine (DIEA)and Diisopropyl Carbodiimide (DIC) were purchased from Sigma-Aldrich.

Synthesis of pMethylbenzyl Seleno-Cysteine

Seleno-Cystine.2 HCl (31.2 mmoles, 10.6 g) was mixed with about 100 mLof 0.5 N NaOH to make a slurry. A solution of Sodium Borohydride (10 g,253 mmoles) in water (60 mL) was added dropwise to the well-stirredsolution of Seleno-Cystine in a 500 mL round bottom flask, which waswell chilled to prevent boiling. After the vigorous reaction, thesolution turns from yellow to colorless. Glacial acetic acid was addeduntil the pH was approximately 6 (˜20 mL). 2-Methylbenzyl Bromide (8.5mL, 63 mmoles) was added dropwise to the above solution. The reactionwas completed in about 30 minutes. The solution was acidified withconcentrated HCl to pH 3 to complete the formation of the precipitate.The precipitate was filtered after letting it stand overnight and wasdissolved in 200 mL boiling water. After letting it stand in therefrigerator for 3 hours, the crystals were filtered, washed with waterand then dried. The yield was 10 g.

Synthesis of Boc-Se (pMB)-Cysteine

pMethylbenzyl (pMB) Seleno-Cysteine. HCl (9 g, 33.3 mmoles) was mixedwith water (90 mL) to make a slurry in a one-liter flask. After addingtriethylamine (TEA) (4.7 mL, 33.3 mmoles) to the slurry at roomtemperature while stirring, a solution of (Boc)_(2O) or di-tert-butylcarbonate (14.5 g, 66.6 mmoles) in acetonitrile (100 mL) was added andan additional amount of TEA (4.7 mL, 33.3 mmoles) was added. Thesolution became clear. The reaction was stirred at room temperature foran additional hour, after which it was acidified with 1 N HCl (45 ML).It was then extracted with ethyl acetate. The ethyl acetate extract waswashed with 1 N HCl (3×100 mL) and the aqueous layer was again extractedwith ethyl acetate. This extract was combined with the first extract,dried over anhydrous Na₂SO₄ and evaporated to dryness using a Rotovap.Yield: 8.0 g

Synthesis of Boc-(methyl)-Seleno-Cysteine

Methyl-Seleno-Cysteine. HCl (6.21 g, 33.3 mmoles) was mixed with water(90 mL) to make a slurry in a one-liter flask. After adding TEA (4.7 mL,33.3 mmoles) to it, at room temperature with good stirring, a solutionof (Boc)_(2O) or di-tert-butyl carbonate (14.5 g, 66.6 mmoles) inacetonitrile (100 mL) was added to it and an additional amount of TEA(4.7 mL, 33.3 mmoles) was added. The solution became clear. The reactionwas stirred at room temperature for an additional hour, after which itwas acidified with 1 N HCl (45 ML). It was then extracted with ethylacetate. The ethyl acetate extract was washed with 1 N HCl (3×100 mL).The aqueous layer was then extracted again with ethyl acetate. Thisextract was combined with the first extract, dried over anhydrous Na₂SO₄and evaporated to dryness using a Rotovap. Yield: 7 g

Synthesis of BeKm-1:

Boc (Tos) Arg¹-Pro-(Bzl) Thr-(Cyh) Asp-Ile-(ClZ) Lys-S (pMB) Cys⁷-(Bzl)Ser-(Cyh) Glu-(Bzl) Ser¹⁰-(BrZ) Tyr-Gln-(pMB) Cys-Phe-Pro¹⁵-Val-(pMB)Cys-(ClZ) Lys-(Bzl) Ser-(Tos) Arg²⁰-Phe-Gly-(ClZ) Lys-(Bzl)Thr-Asn²⁵-Gly-(Tos) Arg-(pMB) Cys-Val-Asn³⁰- Gly-Phe-(pMB) Cys-(Cyh)Asp-(pMB) Cys³⁵-Phe-PAM-resin.

Using Boc protected amino acids and general Boc chemistry the abovesequence was synthesized on Boc-Phe-O-methyl PAM resin. Generally themethod of choice for coupling amino acid residues was using DIC/HOBT.When some amino acids needed to be double coupled, HBTU and DIEA wereused for the second coupling. The completion of couplings was checked bythe Kaiser test.

Synthesis of (SeC)⁷-BeKm-1:

Boc (Tos) Arg¹-Pro-(Bzl) Thr-(Cyh) Asp-Ile-(ClZ) Lys-S(pMB) Cys⁷-(Bzl)Ser-(Cyh) Glu-(Bzl) Ser¹⁰-(BrZ) Tyr-Gln-(pMB) Cys-Phe-Pro ¹⁵-Val-(pMB)Cys-(ClZ) Lys-(Bzl) Ser-(Tos) Arg²⁰-Phe-Gly-(ClZ) Lys-(Bzl) Thr-Asn²⁵-Gly-(Tos) Arg-(pMB) Cys-Val-Asn³⁰-Gly-Phe-(pMB) Cys-(Cyh) Asp-(pMB)Cys³⁵-Phe-PAM-resin

Using Boc protected amino acids and general Boc chemistry the abovesequence was synthesized on Boc-Phe-O-methyl PAM resin. The synthesiswas performed using the same strategy as mentioned above, except for theresidue seven, where Boc-Se (pMB)-Cysteine was used. The same couplingstrategy was used as that used for the BeKm-1 synthesis. The couplingefficiency was checked using the Kaiser test.

Synthesis of (MeSeCys)⁰-BeKm-1

Boc MeSeCys⁰-Boc (Tos) Arg¹-Pro-(Bzl) Thr-(Cyh) Asp-Ile-(ClZ) Lys-(pMB)SeCys⁷-(Bzl) Ser-(Cyh) Glu-(Bzl) Ser¹⁰-(BrZ) Tyr-Gln-(pMB)Cys-Phe-Pro¹⁵-Val-(pMB) Cys-(ClZ) Lys-(Bzl) Ser-(Tos)Arg²⁰-Phe-Gly-(ClZ) Lys-(Bzl) Thr-Asn²⁵-Gly-(Tos) Arg-(pMB)Cys-Val-Asn³⁰- Gly-Phe-(pMB) Cys-(Cyh) Asp-(pMB) Cys³⁵-Phe-PAM-resin

Boc-Phenylalanyl-O-methyl-PAM resin was used with a loading of 0.67mmoles of Boc Phe/gm of resin. The synthesis was performed similar tothe regular BeKm-1 using the same strategy as mentioned above. However,after the completion of the sequence for BeKm-1,Boc-methyl-Seleno-Cysteine was coupled to the residue 1. The samecoupling strategy was used as that used for the BeKm-1 synthesis. Thecoupling efficiency was checked using the Kaiser test.

Method of Cleavage from the Resin and Folding:

For all three peptide resins the following method was used.

i) Removal of Boc Protection:

The resin was initially treated with 50% TFA/DCM to remove the Bocgroup, washed with DCM, DMF and Ether and dried.

ii) HF Cleavage:

The dry resin was transferred to a HF reaction vessel and p-Cresol wasadded (1 ml/g resin). Liquid Hydrogen Fluoride (10 ml/g peptide resin)was added to the reaction vessel, and the reaction was cooled in dryice/acetone and evacuated with high vacuum. The resin was stirred at 0°C. for 1 hour. The HF was then removed at 0° C. under vacuum. The resinwas extracted with a mixture of buffer B (0.1% TFA/ACN) and buffer A(0.1% TFA/H₂O), after washing thoroughly with anhydrous Ether.

iii) Folding:

The peptide solution was diluted with deionized water to 0.5 mM (orpossibly more as long as the percentage of ACN was not less than 10%after dilution), pH was adjusted to 7-7.5 and the solution was stirredat room temperature overnight. The progress of folding was monitored byHPLC. The folding was complete by the next morning. The pH was thenreduced to 4 by adding acetic acid.

iv) Purification:

The solution was centrifuged to remove some insoluble material. Thesolution was then loaded onto a Hamilton PRP-1 column (25×250 mm; 10 mmbeads) using a Rainin Dynamax HPLC machine. The following conditionswere used;

Buffer A: 0.1% TFA/water; Buffer B: 0.1% TFA in acetonitrile, Flow Rate:40 mL/min., Wavelength of detection: 230 nm. Gradient: 10 to 40% B in 80min.The fractions with the right mass were pooled and lyophilized.

The lyophilized peptide was then loaded onto a Phenomenex “Luna (2)”(C18, 10 mm, 100 A, 1″) column with the same buffer system and gradientthat was used earlier but at a flow rate of 9 mL/min and wavelength ofdetection at 230 nm. The analytical runs were done on a Luna (2), 5 mm,100 A, 4.6×250 mm, C18 column on an Agilent 1100 HPLC system, with thesame buffer system at 1 mL/min flow rate, UV 210 nm and gradient 10-40%B in 100 min. The fractions were collected and, based upon theanalytical runs and the mass spectra, the fractions containing the rightmolecular weight material and right purity were pooled and lyophilized.

Flash Blue Competition Assays with BeKm-1 Analogs

Acetate and TFA salts of (SeC)⁷-BeKm-1 were tested in homogeneous[¹²⁵I]-BeKm-1 competition assays using FlashBlue GPCR beads and hERG K⁺channel membranes derived from Perkin Elmer's hERG/HEK-293 cell line(clone 16). Radioligand was used at the K_(d) concentration determinedfor the FlashBlue assay (0.17 nM). Assays were run in buffer composed of20 mM Hepes/Tris pH 7.2, 100 μM KCl, 0.5% BSA. 1.25 μg hERG K⁺ channelmembranes and 62.5 μg FlashBlue GPCR beads were added to each well of a384-well white Optiplate. The total assay volume for each well was 40μl. Plates were spun briefly after a one hour incubation at roomtemperature prior to signal detection with a TopCount apparatus.

The results of the assays are shown in FIG. 4. FIG. 4A is a summary ofK_(i) values of (SeC)⁷-BeKm-1 in comparison to native BeKm-1 andBiotinylated BeKm-1. FIG. 4B is an example of data obtained from aFlashBlue experiment. In FIG. 4B, the closed rectangles show results forbiotinyl-BeKm-1 TFA salt; open triangles are biotinyl-BeKm-1 acetatesalt; stars are (SeC)⁷-BeKm-1 TFA salt; open circles are (SeC)⁷-BeKm-1acetate salt; and open diamonds are BeKm-1 TFA salt. FIG. 4 shows that(SeC)⁷-BeKm-1 analogs have an affinity for the hERG channel comparableto that of native BeKm-1. By comparison, biotinylated analogs of BeKm-1have an affinity that is shifted by about one log to the right.

ICP-MS Quantitation of (SeC)⁷-BeKm-1

ICP-MS analysis was performed, using standard protocols, on a mixture of0.2 mg/L of (SeC)⁷-BeKm-1 in dilute acetic acid solution and 1 mM ofCelebrex in dilute acetate solution at different proportions (%). FIG.5A shows a table and FIG. 5B shows a graph of the results of thisanalysis. FIG. 5 shows that bound competitive inhibitor can be measuredeven when the competitive inhibitor solution only makes up 20% of thetotal volume analyzed. Selenium from the competitive inhibitor can bedetected at a concentration of 0.75 parts per billion, corresponding toa peptide concentration of 7.569 parts per billion.

As one of ordinary skill in the art will appreciate, various changes,substitutions, and alterations could be made or otherwise implementedwithout departing from the principles of the present invention. Forexample, selenium analogs of astemizole, terfenadine, or any other hERGK⁺ channel blocker may be useful according to the present invention.Accordingly, the scope of the invention should be determined by thefollowing claims and their legal equivalents.

1. A composition having the formula:

or a pharmaceutically acceptable salt thereof.
 2. The composition as setforth in claim 1, wherein said pharmaceutically acceptable salt is anacetate, TFA, maleate or hydrochloride salt.
 3. The composition as setforth in claim 1, wherein said composition binds hERG K⁺ channel with aK_(i) in the range of about 4 nM to about 25 nM.
 4. A method ofdetecting binding of a compound to a hERG K⁺ channel, comprising: a)adding said compound to said hERG K⁺ channel in the presence of aselenium analog of a competitive inhibitor of said hERG K⁺ channel; b)quantifying the amount of said selenium analog of said competitiveinhibitor that bound to said hERG K⁺ channel using mass spectrometry;and c) determining the amount of said compound that bound to said hERGK⁺ channel based on said quantifying.
 5. The method as set forth inclaim 4, wherein said selenium analog of said competitive inhibitor isselected from the group consisting of di-seleno-dofelitide,(SeC)⁷-BeKm-1 and (MeSeC)⁰-BeKm-1.
 6. The method as set forth in claim4, wherein said selenium analog of said competitive inhibitor is acombination of di-seleno-dofetilide and (SeC)⁷-BeKm-1 ordi-seleno-dofelitide and (MeSeC)⁰-BeKm-1
 7. The method as set forth inclaim 4, wherein said mass spectrometry is MALDI-TOF mass spectrometry,electrospray mass spectrometry, or ICP mass spectrometry.
 8. The methodas set forth in claim 4, wherein said quantifying comprises quantifyingthe amount of selenium bound to said hERG K⁺ channel.
 9. The method asset forth in claim 4, wherein said selenium analog of said competitiveinhibitor has a K_(i) that is within an order of magnitude of the K_(i)of an unmodified version of said competitive inhibitor.
 10. The methodas set forth in claim 4, wherein said selenium analog of saidcompetitive inhibitor has a K_(i) that is within about 3 times the K_(i)of an unmodified version of said competitive inhibitor.